Chemerin As a New Potential Player in the Immunoregulatory Activity of Mesenchymal Stromal Cells

Although several studies have shown that Mesenchymal Stromal Cells (MSCs) are used for treating inflammatory diseases, the mechanisms underlying their capacity to inhibit the inflammatory response are not understood. Chemerin is an immunoregulatory protein with chemotactic activity, secreted by different cell subsets as a precursor and converted into its active form through the proteolitic cleavage of the last six-seven amino acids at the C-terminal domain.

We showed that MSCs are able to produce Chemerin (MSC-Chem) and its production depends on the conditions used for MSC culture. In particular, we observed that MSC cultured with platelet lysate produced high amount of chemerin under basal conditions, and its secretion was strongly increased after stimulation with inflammatory cytokines. Therefore, in order to understand if MSC-Chem is involved in the immunoregulatory function of MSCs, we performed biochemical and functional analysis. To evaluate the chemotactic activity of MSC-Chem, we performed migration assays using a pre-B cell line expressing the human ChemR23 receptor (L1.2-ChemR23). L1.2-ChemR23 cells were able to migrate in response to rh-chemerin in a dose depend manner, until the concentration of 5nM (at 0.2 nM MI=2472, range=2201-2743; at 1 nM MI=9392, range=8902-9882; at 5nM MI=11737, range=11665-11809, at 10 nM MI=2904, range=3261-2548; p= 0.01). Interestingly, MSC-Chem induced the migration of L1.2-ChemR23 cells at 1 nM, 5 nM and 10nM (MI=85, 480, 1131, respectively). However, at equivalent concentrations, rh-chemerin was able to induce a stronger L1.2-ChemR23 migration compared to MSC-Chem, suggesting that within MSCs supernatant only a fraction of the protein was in the active form. In accordance, the biochemical analysis obtained by the LC/MS mass spectrometry identified chemerin active form (with the last peptide Chem₁₄₄-Chem₁₄₇) only in rh-chemerin, but not in MSC-Chem (Chem₁₄₄-Chem₁₄₇), confirming that most of the MSC-Chem was in the inactive form.

Chemerin has been reported to be cleaved by several serine and cysteine proteases, which are then able to activate or inactivate chemerin, depending on the cleavage site. We analysed the expression of chemerin serine-cysteine proteases by MSCs both under basal conditions and after stimulation with inflammatory cytokines. RT-PCR showed that MSC express low levels of neutrophil elastase (mean 2^-ΔΔCt=1, range=0.55-1.4 n=3) compared to PBMCs (positive control) (mean 2^-ΔΔCt=234.5, range=201.3-284.7 n=3) and its expression did not significantly increase after 24h, 48h or 72h of stimulation with inflammatory cytokines (mean 2^-ΔΔCt = 5, range=3.1-5.8; mean 2^-ΔΔCt = 3.0, range=2.6 -3.7; mean 2^-ΔΔCt = 2,1, range= 1.9-2.4; respectively, n=3). MSCs also expressed cathepsin K (mean 2^-ΔΔCt = 7.4, range= 4.4-10.2, n=2), and its levels did not increase after stimulation with inflammatory cytokines (after 24h mean 2^-ΔΔCt = 8.5, range= 3.75-19.1; after 48h mean 2^-ΔΔCt = 8.8, range=5.0-12.6 and after 72h mean 2^-ΔΔCt= 10.5, range=9.4-11,8; n=2).

In conclusion, we demonstrated that MSCs were able to produce Chemerin and directly cleave it in its active form, although only partially. We speculate that, when infused in vivo during inflammation, MSCs produce chemerin as a precursor, which is then converted in its active form by cysteine and serine proteases, highly expressed at peripheral inflamed tissues. Further in vivo studies are needed to show whether activated Chemerin can then induce ChemR23-expressing cells migration towards MSC, to better exert their anti-inflammatory activity.